Restoration of pathogenicity of avirulent Xanthomonas oryzae pv. oryzae and X. campestris pathovars by reciprocal complementation with the hrpXo and hrpXc genes and identification of HrpX function by sequence analyses.

The molecular basis of pathogenesis by Xanthomonas oryzae pv. oryzae has been partly elucidated by the identification of a gene, hrpXo, required for bacterial blight on rice. A mutation in hrpXo results in the loss of pathogenicity on rice and the loss of hypersensitivity on nonhosts such as Datura stramonium and radishes. Pathogenicity and its ability to cause the hypersensitive reaction is restored by complementing the mutant with the heterologous hrpXc gene derived from X. campestris pv. campestris. Conversely, hrpXo complements nonpathogenic mutants of X. campestris pv. campestris and X. campetstris pv, armoraciae. Mutants bearing the heterologous hrpX gene are restored in their abilities to cause diseases typical of their chromosomal background and not the hypersensitive reaction on their respective hosts. The hrpXo and hrpXc genes are therefore functionally equivalent, and this functional equivalence extends into X. campestris pv. armoraciae and possibly into other X. campestris pathovars, since this gene is highly conserved among eight other pathovars tested. Sequence analyses of hrpXo revealed an open reading frame of 1,452 bp with a coding capacity for a protein of 52.3 kDa. The protein contains a consensus domain for possible protein myristoylation whose consequence may result in a loss of recognition by host defense and surveillance systems.

Pseudomonas syringae pv. phaseolicola genomic clones harboring heterologous DNA sequences suppress the same phaseolotoxin-deficient mutants.

Cosmid cloning and mutagenesis were used to identify genes involved in the production of phaseolotoxin, the chlorosis-inducing phytotoxin of Pseudomonas syringae pv. phaseolicola, the causal agent of halo blight of bean (Phaseolus vulgaris L.). Eight stable clones were isolated from a genomic cosmid library by en masse mating to 10 ethyl methanesulfonate (EMS)-induced Tox- mutants. In cross-matings, each suppressed all 10 mutants as well as an additional 70 EMS-induced Tox- mutants (and one UV-induced Tox- mutant). On the basis of restriction endonuclease analysis and hybridization studies, the clones were grouped into three classes. Clones in a particular class shared common fragments, whereas clones in different classes did not. Clones from class I (but not classes II and III) also suppressed Tn5-induced Tox- mutants. Interposon mutagenesis and marker exchange of a representative clone from class III into the wild-type genome did not alter its Tox+ phenotype, indicating that this clone does not harbor structural or regulatory genes involved in phaseolotoxin production. We suggest that the genome of P. syringae pv. phaseolicola contains a "hot spot" in one of the functions involved in toxin production which is affected by EMS and UV and that heterologous clones are able to suppress the Tox- phenotype because their inserts encode products that are able to substitute for the product of the mutated gene. Alternatively, the inserts may contain sequences which titrate a repressor protein. In either case, the data suggest that suppression of EMS- and UV-induced mutants occurs when heterologous clones are present in multiple copies.